Catecholate Siderophore Research Articles

In vivo growth of Staphylococcus lugdunensis is facilitated by the concerted function of heme and non-heme iron acquisition mechanisms

Staphylococcus lugdunensis has increasingly been recognized as a pathogen that can cause serious infection indicating this bacterium overcomes host nutritional immunity. Despite this, there exists a significant knowledge gap regarding the iron acquisition mechanisms employed by S. lugdunensis, especially during infection of the mammalian host. Here we show that S. lugdunensis can usurp hydroxamate siderophores and staphyloferrin A and B from Staphylococcus aureus. These transport activities all required a functional FhuC ATPase. Moreover, we show that the acquisition of catechol siderophores and catecholamine stress hormones by S. lugdunensis required the presence of the sst-1 transporter-encoding locus, but not the sst-2 locus. Iron-dependent growth in acidic culture conditions necessitated the ferrous iron transport system encoded by feoAB. Heme iron was acquired via expression of the iron-regulated surface determinant (isd) locus. During systemic infection of mice, we demonstrated that while S. lugdunensis does not cause overt illness, it does colonize and proliferate to high numbers in the kidneys. By combining mutations in the various iron acquisition loci (isd, fhuC, sst-1, and feo), we demonstrate that only a strain deficient for all of these systems was attenuated in its ability to proliferate to high numbers in the murine kidney. We propose the concerted action of heme and non-heme iron acquisition systems also enable S. lugdunensis to cause human infection.

Ruckerbactin Produced by Yersinia ruckeri YRB Is a Diastereomer of the Siderophore Trivanchrobactin Produced by Vibrio campbellii DS40M4.

The Gram-negative bacterium Yersinia ruckeri is the causative agent for enteric red mouth disease in salmonids. The genome of Y. ruckeri YRB contains a biosynthetic gene cluster encoding the biosynthesis of catechol siderophores that are diastereomeric with the known vanchrobactin class of siderophores, (DHBDArgLSer)(1-3). Ruckerbactin (1), produced by Y. ruckeri YRB, was found to be the linear tris-l-serine ester composed of l-arginine and 2,3-dihydroxybenzoic acid, (DHBLArgLSer)3. The biscatechol, (DHBLArgLSer)2 (2), and monocatechol, DHBLArgLSer (3), compounds were also isolated and characterized. The macrolactone of ruckerbactin was not detected. The presence of LArg in ruckerbactin makes it the diastereomer of trivanchrobactin with DArg. The electronic circular dichroism spectra of Fe(III)-ruckerbactin and Fe(III)-trivanchrobactin reveal the opposite enantiomeric configurations at the Fe(III) sites. Fe(III)-ruckerbactin adopts the Δ configuration, and Fe(III)-trivanchrobactin adopts the Λ configuration. Y. ruckeri YRB was also found to produce the antimicrobial agent holomycin (4).

Iron-Utilization System in Vibrio vulnificus M2799.

Vibrio vulnificus is a Gram-negative pathogenic bacterium that causes serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In the ferric-utilization system in V. vulnificus M2799, an isochorismate synthase (ICS) and an outer membrane receptor, VuuA, are required under low-iron conditions, but alternative proteins FatB and VuuB can function as a periplasmic-binding protein and a ferric-chelate reductase, respectively. The vulnibactin-export system is assembled from TolCV1 and several RND proteins, including VV1_1681. In heme acquisition, HupA and HvtA serve as specific outer membrane receptors and HupB is a sole periplasmic-binding protein, unlike FatB in the ferric-vulnibactin utilization system. We propose that ferric-siderophore periplasmic-binding proteins and ferric-chelate reductases are potential targets for drug discovery in infectious diseases.

Antifungal Activity of Siderophore Isolated From Escherichia coli Against Aspergillus nidulans via Iron-Mediated Oxidative Stress.

Microorganisms produce various secondary metabolites for growth and survival. During iron stress, they produce secondary metabolites termed siderophores. In the current investigation, antifungal activity of catecholate siderophore produced by Escherichia coli has been assessed against Aspergillus nidulans. Exogenous application of the bacterial siderophore to fungal cultures resulted in decreased colony size, increased filament length, and changes in hyphal branching pattern. Growth inhibition was accompanied with increased intracellular iron content. Scanning electron microscopy revealed dose-dependent alteration in fungal morphology. Fluorescent staining by propidium iodide revealed cell death in concert with growth inhibition with increasing siderophore concentration. Antioxidative enzyme activity was also compromised with significant increase in catalase activity and decrease in ascorbate peroxidase activity. Siderophore-treated cultures showed increased accumulation of reactive oxygen species as observed by fluorescence microscopy and enhanced membrane damage in terms of malondialdehyde content. Antifungal property might thus be attributed to xenosiderophore-mediated iron uptake leading to cell death. STRING analysis showed interaction of MirB (involved in transport of hydroxamate siderophore) and MirA (involved in transport of catecholate siderophore), confirming the possibility of uptake of iron–xenosiderophore complex through fungal transporters. MirA structure was modeled and validated with 95% residues occurring in the allowed region. In silico analysis revealed MirA–Enterobactin–Fe3+ complex formation. Thus, the present study reveals a promising antifungal agent in the form of catecholate siderophore and supports involvement of MirA fungal receptors in xenosiderophore uptake.

Radiochemical Evidence for the Contribution of Chemotyped Siderophore Producing Bacteria Towards Plant Iron Nutrition.

Fe deficiency is a major challenge that limits agricultural productivity and is a serious human health concern worldwide. Under iron-limiting conditions soil microorganisms produce siderophores, that chelates Fe3+ (ferric) and make it available to the plants. Selection of efficient siderophore producing bacteria and establishing their role in enhancing iron uptake is a strategic approach for improving plant nutrition. Hence 3 efficient isolates Pantoea agglomerans, Pseudomonas plecoglossida and Lactococcus lactis, selected from a repository of 154 bacteria, producing catecholate, hydroxamate and carboxylate siderophores, respectively, were assessed for Fe chelation efficiency using 59Fe and their role in plant biometric parameters, Fe uptake and antioxidant enzymes with tomato (Strategy I) and wheat (Strategy II) test plants under hydroponic system. Cell-free siderophore preparation (Sid) improved plant parameters and iron nutrition more efficiently than bacterial inoculants. Pantoea agglomerans was proven best as its 59Fe-bound siderophore complex showed the highest uptake of 4.25 and 1.59Bq plant-1 in wheat and tomato, respectively. Further, the Fe-starved plants (1µm Fe-EDTA) showed around two-fold higher 59Fe uptake than those raised under Fe-sufficient condition (100µm Fe-EDTA). Results indicated that probably the bacterial mediated iron translocation in plants is Strategy III, complementing both Strategy I and II by facilitating higher availability of chelated Fe to plant reductases directly and/or through ligand exchange with phytosiderophores, respectively. This study highlights the need for integration of siderophore based formulations in INM strategies for enhancing plant iron content to address the Fe deficiency challenge of the soil and human nutrition.

Production, Purification, and Characterization of Bacillibactin Siderophore of Bacillus subtilis and Its Application for Improvement in Plant Growth and Oil Content in Sesame

Siderophores are low molecular weight secondary metabolites produced by microorganisms under low iron stress as a specific iron chelator. In the present study, a rhizospheric bacterium was isolated from the rhizosphere of sesame plants from Salem district, Tamil Nadu, India and later identified as Bacillus subtilis LSBS2. It exhibited multiple plant-growth-promoting (PGP) traits such as hydrogen cyanide (HCN), ammonia, and indole acetic acid (IAA), and solubilized phosphate. The chrome azurol sulphonate (CAS) agar plate assay was used to screen the siderophore production of LSBS2 and quantitatively the isolate produced 296 mg/L of siderophores in succinic acid medium. Further characterization of the siderophore revealed that the isolate produced catecholate siderophore bacillibactin. A pot culture experiment was used to explore the effect of LSBS2 and its siderophore in promoting iron absorption and plant growth of Sesamum indicum L. Data from the present study revealed that the multifarious Bacillus sp. LSBS2 could be exploited as a potential bioinoculant for growth and yield improvement in S. indicum.

Enterobactin- and salmochelin-β-lactam conjugates induce cell morphologies consistent with inhibition of penicillin-binding proteins in uropathogenic Escherichia coli CFT073.

The design and synthesis of narrow-spectrum antibiotics that target a specific bacterial strain, species, or group of species is a promising strategy for treating bacterial infections when the causative agent is known. In this work, we report the synthesis and evaluation of four new siderophore-β-lactam conjugates where the broad-spectrum β-lactam antibiotics cephalexin (Lex) and meropenem (Mem) are covalently attached to either enterobactin (Ent) or diglucosylated Ent (DGE) via a stable polyethylene glycol (PEG3) linker. These siderophore-β-lactam conjugates showed enhanced minimum inhibitory concentrations against Escherichia coli compared to the parent antibiotics. Uptake studies with uropathogenic E. coli CFT073 demonstrated that the DGE-β-lactams target the pathogen-associated catecholate siderophore receptor IroN. A comparative analysis of siderophore-β-lactams harboring ampicillin (Amp), Lex and Mem indicated that the DGE-Mem conjugate is advantageous because it targets IroN and exhibits low minimum inhibitory concentrations, fast time-kill kinetics, and enhanced stability to serine β-lactamases. Phase-contrast and fluorescence imaging of E. coli treated with the siderophore-β-lactam conjugates revealed cellular morphologies consistent with the inhibition of penicillin-binding proteins PBP3 (Ent/DGE-Amp/Lex) and PBP2 (Ent/DGE-Mem). Overall, this work illuminates the uptake and cell-killing activity of Ent- and DGE-β-lactam conjugates against E. coli and supports that native siderophore scaffolds provide the opportunity for narrowing the activity spectrum of antibiotics in clinical use and targeting pathogenicity.

VuuB and IutB reduce ferric-vulnibactin in Vibrio vulnificus M2799.

Vibrio vulnificus, a pathogenic bacterium that causes serious infections in humans, requires iron for growth. Clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, namely, vulnibactin, to capture iron (III) from the environment. Growth experiments using a deletion mutant indicated that VuuB, a member of the FAD-containing siderophore-interacting protein family, plays a crucial role in Fe3+-vulnibactin reduction. IutB, a member of the ferric-siderophore reductase family, stands a substitute for VuuB in its absence. It remained unclear why V. vulnificus M2799 has two proteins with relevant functions. Here we biochemically characterized VuuB and IutB using purified recombinant proteins. Purified VuuB, a flavoprotein, catalyzed the reduction of Fe3+-nitrilotriacetic acid as its electron acceptor, in the presence of NADH as its electron donor and FAD as its cofactor. IutB catalyzed the reduction of Fe3+-nitrilotriacetic acid, in the presence of NADH, NADPH, or reduced glutathione as its electron donor. The optimal pH values and temperatures of VuuB and IutB were 7.0 and 37°C, and 8.5 and 45°C, respectively. On analyzing their ferric-chelate reductase activities, both VuuB and IutB were found to catalyze the reduction of Fe3+-aerobactin, Fe3+-vibriobactin, and Fe3+-vulnibactin. When the biologically relevant substrate, Fe3+-vulnibactin, was used, the levels of ferric-chelate reductase activities were similar between VuuB and IutB. Finally, the mRNA levels were quantified by qRT-PCR in M2799 cells cultivated under low-iron conditions. The number of vuuB mRNA was 8.5 times greater than that of iutB. The expression ratio correlated with the growth of their mutants in the presence of vulnibactin.

Case Report: Successful Rescue Therapy of Extensively Drug-Resistant Acinetobacter baumannii Osteomyelitis With Cefiderocol.

Cefiderocol is a novel catechol siderophore cephalosporin antibiotic developed to treat resistant gram-negative infections. We describe its successful use as rescue therapy, combined with surgical debridement, to treat a patient with osteomyelitis due to extensively drug-resistant Acinetobacter baumannii. Bacterial whole-genome sequencing identified the strain and antibiotic resistance determinants.

Transport of Degraded Ferric Enterobactin by Fiu

Under iron deficient conditions, Gram‐negative bacteria like Escherichia coli secrete enterobactin, a catecholate siderophore that complexes and solubilizes iron in the extracellular environment. Ferric enterobactin (FeEnt) is the most avid natural or synthetic iron complex. It may be glucosylated (FeGEnt), and it is also subject to both chemical degradation and oxidation (FeEnt*). Neverthless, bacterial outer membrane transporters acquire FeEnt, FeGEnt and FeEnt*. For example, the E. coli Fiu protein is known to catalyze the uptake of monomeric catecholate iron complexes, and using siderophore nutrition tests and radioisotopic iron accumulation experiments, we found that Fiu preferentially recognizes and transports FeEnt*. To further analyze the specificity of the FeEnt* uptake process, we created Cys substitutions in the external surface loops of Fiu and labeled them with fluorescein maleimide (FM). During binding of ferric siderophores, the surface loops undergo conformational motion that quenches attached fluorophores, allowing quantitative determinations of affinity and specificity. We measured the affinity of Fiu‐FM for different ferric catecholate compounds: FeEnt*, FeEnt, and FeGEnt produced Kd values of 0.46, 20, and 0.5 μM, respectively. Thus, Fiu manifests specificity for the degradation products of FeEnt, but also mediates the uptake of FeEnt and FeGEnt, identifying it as an important component of bacterial iron acquisition systems that are essential to bacterial survival.Support or Funding InformationNational Institutes of Health

EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058

Extraintestinal pathogenic Escherichia coli (ExPEC) shows an enhanced ability to cause infection outside the intestinal tract. Avian pathogenic E. coli (APEC), one type of ExPEC, causes avian colibacillosis, a disease of significant economic importance to poultry producers worldwide that is characterized by systemic infection. Some ExPEC strains as well as other pathogenic enterobacteria produce enterobactin, a catecholate siderophore used to sequester iron during infection. Here, we showed that disruption of enterobactin efflux via outer membrane protein TolC significantly decreased the pathogenicity of APEC strain E058. Furthermore, colonization and persistence assays performed using a chicken infection model showed that the ΔtolC mutant was obviously attenuated (p˂0.001). In contrast, disruption of enterobactin synthesis gene entE and/or the inner membrane transporter gene entS had little effect on pathogenicity. Analysis of growth kinetics revealed a significant reduction in the growth of triple mutant strain E058ΔentEΔentSΔtolC in iron-deficient medium compared with the wild-type strain (p˂0.001), while no growth impairment was noted for the E058ΔtolC mutant in either Luria-Bertani broth or iron-deficient medium. The E058ΔentEΔentSΔtolC mutant also showed significantly decreased virulence compared with single mutant strain E058ΔtolC. Low-copy complementation of strains E058ΔtolC and E058ΔentEΔentSΔtolC with plasmid-borne tolC restored virulence to wild-type levels in the chicken infection model. Macrophage infection assays showed that ingestion of E058ΔtolC by macrophage cell line HD11 cells was reduced compared with ingestion of the E058ΔentEΔentSΔtolC mutant. However, no significant differences were observed between the mutants and the wild-type in a chicken serum resistance assay. Together, these results suggest that EntE, EntS and TolC synergistically contributed to the pathogenesis of APEC strain E058 in an iron-deficient environment.

Precursor-directed biosynthesis of catechol compounds in Acinetobacter bouvetii DSM 14964.

Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin (1), butanochelin (2), and pentanochelin (3). This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(iii) and may be further modified via thiol-ene or azide-alkyne click chemistry.

A Microbial Siderophore-Inspired Self-Gelling Hydrogel for Noninvasive Anticancer Phototherapy.

Microbial carboxyl and catechol siderophores have been shown to have natural iron-chelating abilities, suggesting that hyaluronic acid (HA) and the catechol compound, gallic acid (GA), may have iron-coordinating activities. Here, a photoresponsive self-gelling hydrogel that was both injectable and could be applied to the skin was developed on the basis of the abilities of HA and GA to form coordination bonds with ferric ions (Fe3+). The conjugate of HA and GA (HA-GA) instantly formed hydrogels in the presence of ferric ions and showed near-infrared (NIR)-responsive photothermal properties. Following their subcutaneous injection into mice, HA-GA and ferric ion formed a hydrogel, which remained at the injection site for at least 8 days. Intratumoral injection of HA-GA/Fe hydrogel into mice allowed repeated exposure of the tumor to NIR irradiation. This repeated NIR irradiation resulted in complete tumor ablation in KB carcinoma cell-xenografted mice and suppressed lung metastasis of 4T1-Luc orthotopic breast tumors. Application of HA-GA/Fe hydrogel to the skin of A375 melanoma-xenografted tumor sites, followed by NIR irradiation, also resulted in complete tumor ablation. These findings demonstrate that single applications of HA-GA/Fe hydrogel have photothermal anticancer effects against both solid tumors and skin cancers. SIGNIFICANCE: These findings provide new insights into noninvasive anticancer phototherapy using self-gelling hydrogels. Application of these hydrogels in preclinical models reduces the sizes of solid tumors and skin cancers without surgery, radiation, or chemotherapy.

Impaired Efflux of the Siderophore Enterobactin Induces Envelope Stress in Escherichia coli.

The Cpx response is one of several envelope stress responses that monitor and maintain the integrity of the gram-negative bacterial envelope. While several conditions that are known or predicted to generate misfolded inner membrane proteins activate the Cpx response, the molecular nature of the Cpx inducing cue is not yet known. Studies have demonstrated that mutation of multidrug efflux pumps activates the Cpx response in many gram-negative bacteria. In Vibrio cholerae, pathway activation is due to accumulation of the catechol siderophore vibriobactin. However, the mechanism by which the Cpx response is activated by mutation of efflux pumps in Escherichia coli remains unknown. Here we show that inhibition of efflux by deletion of tolC, the outer membrane channel of several multidrug efflux pumps, activates the Cpx response in E. coli as a result of impaired efflux of the siderophore enterobactin. Enterobactin accumulation in the tolC mutant reduces activity of the nicotinamide adenine dinucleotide (NADH) oxidation arm of the aerobic respiratory chain. However, the Cpx pathway remains active in the tolC mutant when either NADH dehydrogenase I, NADH dehydrogenase II, or cytochrome bo3 is absent. Finally, we show that the Cpx response down-regulates transcription of the enterobactin biosynthesis operon. These results suggest that the Cpx response promotes adaptation to envelope stress in enteric bacteria that are exposed to iron-limited environments, which are rich in envelope-damaging compounds and conditions.

The structure of the bacterial iron–catecholate transporter Fiu suggests that it imports substrates via a two-step mechanism

The ferric iron uptake (Fiu) transporter from Escherichia coli functions in the transport of iron–catecholate complexes across the bacterial outer membrane, providing the bacterium with iron, which is essential for growth. Recently it has become clear that Fiu also represents a liability for E. coli because its activity allows import of antimicrobial compounds that mimic catecholate. This inadvertent import suggests the potential utility of antimicrobial catechol siderophore mimetics in managing bacterial infections. However, to fully exploit these compounds, a detailed understanding of the mechanism of transport through Fiu and related transporters is required. To address this question, we determined the crystal structure of Fiu at 2.1–2.9 Å and analyzed its function in E. coli. Through analysis of the Fiuo crystal structure, in combination with in silico docking and mutagenesis, we provide insight into how Fiu and related transporters bind catecholate in a surface-exposed cavity. Moreover, through determination of the structure of Fiu in multiple crystal states, we revealed the presence of a large, selectively gated cavity in the interior of this transporter. This chamber is large enough to accommodate the Fiu substrate and may allow import of substrates via a two-step mechanism. This would avoid channel formation through the transporter and inadvertent import of toxic molecules. As Fiu and its homologs are the targets of substrate-mimicking antibiotics, these results may assist in the development of these compounds.

The Outer Membrane Protein FstC of Aeromonas salmonicida subsp. salmonicida Acts as Receptor for Amonabactin Siderophores and Displays a Wide Ligand Plasticity. Structure-Activity Relationships of Synthetic Amonabactin Analogues.

Amonabactins are a group of four related catecholate siderophores produced by several species of the genus Aeromonas, including A. hydrophila and the fish pathogen A. salmonicida subsp. salmonicida. Although the gene cluster encoding amonabactin biosynthesis also contains a gene that could encode the ferri-siderophore receptor (fstC), to date there is no experimental evidence to explain its role. In this work, we report the identification of the amonabactins' outer membrane receptor and the determination of the minimal structural parts of these siderophores involved in the molecular recognition by their cognate receptor. The four natural amonabactin forms (P750, T789, P693, and T732) and some mono and biscatecholate amonabactin analogues were chemically synthesized, and their siderophore activity on A. salmonicida FstC(+) and FstC(-) strains was evaluated. The results showed that each amonabactin form has quite different growth promotion activity, with P750 and T789 the most active. The outer membrane receptor FstC recognizes more efficiently biscatecholate siderophores in which the length of the linker between the two iron-binding catecholamide units is 15 atoms (P750 and T789) instead of 12 atoms (P693 and T732). Analysis of the siderophore activity of synthetic analogues indicated that the presence of Phe or Trp residues is not required for siderophore recognition. The results together point toward evidence that the amonabactin receptor FstC admits a high degree of ligand plasticity. We also showed that FstC is present in most Aeromonas species, including relevant human and animal pathogens as A. hydrophila. From the results obtained, we concluded that the ferri-amonabactin uptake pathway involving the outer membrane transporter FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. This would allow the use of the siderophore-based iron uptake mechanisms to combat infections caused by species of the genus Aeromonas.

An effective approach of bacterial siderophore as nitrogen source triggering the desired biochemical changes in microalgae Chlorella variabilis ATCC 12198

The present study was aimed to check whether catecholate siderophore can serve as a nitrogen source besides iron for microalgae. Catecholate siderophore from Idiomarina loihiensis RS 14 contains 13% nitrogen which can be a potential nitrogen source. Bacterial supernatant containing siderophores with different inoculum ratio was used to check the growth of Chlorella variabilis ATCC 12198. Nitrogen starvation with two stage cultivation was employed here to trigger the accumulation of lipid and carbohydrate. In continuous cultivation, the highest productivity of DCW was observed in 1%, 760 mg/L compared to culture grown at 0.5%, with an increase of 42.2% higher biomass productivity (21.66 mg/L/day). Similarly, in 1%, C. variabilis produced total 30% of lipid content which contains 75% of neutral lipid. Highest carbohydrate was (49.29%) found in cells grown under 9 days of stress in 1% followed by cells grown under 3 days of stress in 5% (46.52%) in stage II cultivation.

Deciphering multifarious properties of Pseudomonads suppressing tomato (Solanum lycopersicum) wilt

Wilt is the most common disease encountered in tomato crop. To devise an eco-friendly management module against F. oxysporum f. spp. solani, the causal organism, native isolates of Pseudomonas spp. were isolated from agricultural fields. The present experiment was conducted at SKUAST-Jammu between 2014-15. Biochemical screening of isolates revealed that antimicrobial compounds, viz. 2,4-DAPG and siderophore production were maximum in isolate PS 1 (217. 70 μg/ml and 32. 25 μg/ml) and HCN production was highest in isolate PS 6 (0. 11 μg/ml). Isolates PS 1 to PS 20 produced ferrate hydroxamate type siderophores whereas PS 21 to PS 30 produced catecholate type siderophores. Growth promoting substances, viz. IAA, GA and phosphorus solubilization were maximum in PS 7 (583. 63 μg/ml), PS 15 (652. 07 μg/ml) and PS 30 (358. 25 μg/ml) respectively. TLC of 2,4-DAPG extracted from various isolates showed Rf values in the range of 0. 7 to 0. 89, close to the relative mobilty (Rf) of phloroglucinol (0. 89). PS 1 reported highest seed germination (75%) with vigour index of 1275. Pot experiment revealed that isolate PS 4 increased plant height, plant growth parameters and further reduced wilt incidence (34. 25%) as compared to the seedling treated with Fusarium oxysporum (60%).

Evaluation of The Pathogenic Potential of Insecticidal Serratia marcescens Strains to Humans.

We observed the death of insect caterpillars of Spodoptera exigua in the laboratory culture line and identified Serratia marcescens as the bacterial causative agent of the insect death. We confirmed that S. marcescens had insecticidal activity against S. exigua and caused high mortality of larvae. The LC50 values of S. marcescens CFU per 1 cm2 of insect diet surface were similar for all isolates. Our research reports novel strains with high pesticidal activity as candidates for future research on a new bioinsecticide. As bioinsecticides cannot be harmful to non-target organisms, we determined the pathogenic properties of S. marcescens to humans. We proved the ability of S. marcescens to damage mammalian epithelial cells. All strains had cytopathic effects to Vero cells with a cytotoxic index ranging from 51.2% ± 3.8% to 79.2% ± 4.1%. We found that all of the strains excreted catecholate siderophore - enterobactin. All isolates were resistant to sulfamethoxazole, tobramycin, gentamicin, cefepime, and aztreonam. We did not observe the ESBL phenotype and the integrons' integrase genes. Resistance to sulfamethoxazole was due to the presence of the sul1 or sul2 gene. The use of resistant S. marcescens strains that are pathogenic to humans in plant protection may cause infections difficult to cure and lead to the spread of resistance genes. The results of our study emphasize the necessity of determination of the safety to vertebrates of the bacteria that are proposed to serve as biocontrol agents. The novelty of our study lies in the demonstration of the indispensability of the bacteria verification towards the lack of hazardous properties to humans.We observed the death of insect caterpillars of Spodoptera exigua in the laboratory culture line and identified Serratia marcescens as the bacterial causative agent of the insect death. We confirmed that S. marcescens had insecticidal activity against S. exigua and caused high mortality of larvae. The LC50 values of S. marcescens CFU per 1 cm2 of insect diet surface were similar for all isolates. Our research reports novel strains with high pesticidal activity as candidates for future research on a new bioinsecticide. As bioinsecticides cannot be harmful to non-target organisms, we determined the pathogenic properties of S. marcescens to humans. We proved the ability of S. marcescens to damage mammalian epithelial cells. All strains had cytopathic effects to Vero cells with a cytotoxic index ranging from 51.2% ± 3.8% to 79.2% ± 4.1%. We found that all of the strains excreted catecholate siderophore – enterobactin. All isolates were resistant to sulfamethoxazole, tobramycin, gentamicin, cefepime, and aztreonam. We did not observe the ESBL phenotype and the integrons’ integrase genes. Resistance to sulfamethoxazole was due to the presence of the sul1 or sul2 gene. The use of resistant S. marcescens strains that are pathogenic to humans in plant protection may cause infections difficult to cure and lead to the spread of resistance genes. The results of our study emphasize the necessity of determination of the safety to vertebrates of the bacteria that are proposed to serve as biocontrol agents. The novelty of our study lies in the demonstration of the indispensability of the bacteria verification towards the lack of hazardous properties to humans.

Growth of Salmonella enterica Serovars Typhimurium and Enteritidis in Iron-Poor Media and in Meat: Role of Catecholate and Hydroxamate Siderophore Transporters

Enteritidis and Typhimurium are among the top Salmonella enterica serovars implicated in human salmonellosis worldwide. This study examined the individual and combined roles of catecholate-iron and hydroxamate-iron transporters in the survival in meat of Salmonella Enteritidis and Typhimurium. Catecholate-iron-III (Fe3+) and hydroxamate-Fe3+ transporter genes fepA, iroN, and fhuACDB were deleted in isolates of these serovars to generate single, double, and triple mutants. Growth rate in high- and low-iron media was compared among mutants, complements, and their wild-type parents. Susceptibility to 14 antibiotics, the ability to produce and utilize siderophores, and survival on cooked chicken breast were evaluated. In iron-poor liquid media, differences were observed between the growth characteristics of mutant Salmonella Enteritidis and Typhimurium. The double Δ iroNΔ fepA and the triple Δ fhuΔ iroNΔ fepA mutants of Salmonella Enteritidis exhibited prolonged lag phases (λ = 9.72 and 9.53 h) and a slow growth rate (μmax = 0.35 and 0.25 h-1) similar to that of its Δ tonB mutant (λ = 10.12 h and μmax = 0.30 h-1). In Salmonella Typhimurium, double Δ iroNΔ fepA and triple Δ fhuΔ iroNΔ fepA mutations induced a similar growth pattern as its Δ tonB mutant. Double deletions of fepA and iroN reduced the siderophore production and the use of enterobactin as an iron source. In the Δ iroNΔ fepA mutant, but not in Δ fhuΔ iroNΔ fepA, the ferrichrome or deferrioxamine promoted growth for both serovars, confirming the specific role of the FhuACDB system in the uptake and transport of hydroxamate Fe3+. Survival of the mutants was also evaluated in a meat assay, and no difference in survival was observed among the mutants compared with wild type. This study showed differences between serovars in the importance of catecholate-iron and hydroxamate-iron uptake on Salmonella growth in iron-restricted media. Data also confirmed that both Salmonella Enteritidis and Typhimurium are well equipped to survive on cooked chicken meat, offering a rich iron condition.